Method of screening of substances for their effect on the expression of mediators of inflammation in a hair follicle

ABSTRACT

An assay for determining whether a first substance is potentially suitable for use as an active agent in hair treating compositions is provided. This assay involves contacting a first substance having putative activity as an active agent for treating hair with plucked hair follicle contained in a culture medium and assaying for a second substance which is correlated to the potential efficacy of the first substance. This assay is particularly suitable for use in evaluating the potential efficacy of hair growth promoters, hair growth retardants, agents that affect hair color, agents that affect hair survival and agents that affect inflammation. This assay is especially suited for evaluating putative efficacy of agents for treatment and/or prevention of the inflammatory stages of alopecia. In particular, the compound to be quantitatively determined is a mediator of inflammation.

This application is a continuation of application Ser. No. 08/603,122,filed Feb. 20, 1996 now abandonded.

The subject of the invention is a process for testing a substance, thesaid substance possibly being active in the hair field.

Hair field is understood to mean everything which can relate to the hairof an individual. Thus, "substance which is possibly active in the hairfield", or simply "substance" subsequently in the text, is understood tomean any molecule or collection of molecules exhibiting a potentialactivity in the hair field and in particular any molecule or collectionof molecules potentially having an activity on the colouring, thesurvival, the slowing down or halting of the growth, the loss oralternatively the intensified growth of hair follicles. The substance tobe tested can be used according to the process of the invention eitherin its molecular form or in the form of a composition containing themolecule to be tested.

To date, the prior art mentions two main methods for testing for asubstance which is possibly active in the hair field. The first consistsin carrying out tests on volunteers and observing more or less rapidlythe effects of the tested substance. It is obvious that this method hasmany disadvantages, including in particular that of being applied tohuman beings, which obviously limits, for ethical reasons, the field ofapplication of the method. Thus, the number and the quality of thesubstances tested is limited. Moreover, these tests are generallycumbersome to carry out and extend over lengthy time periods. Theresults of the test are then in the majority of cases observations ofphenotypic modifications of the hair follicle.

The second method known in the prior art is that described in Patent EP434,319. This method consists in carrying out the tests according to aprocess which involves four stages, including in particular a stage ofisolation of a viable hair follicle which has retained an intact bulb.To do this, it proves necessary to carry out a microdissection of thehair follicle, from a sample withdrawn from a subject. The latter in anycase retains a mark of this operation. This limits the supply of hairfollicles and the implementation of the test method is cumbersome andlengthy.

After much work, the Applicant Company has been able to show that it ispossible to use, in the hair field, at least one plucked hair folliclefor testing a substance which is possibly active in the hair field.

The subject of the invention is therefore a process for testing asubstance which is possibly active in the hair field, characterized inthat at least one hair follicle which has been plucked from a subject isincubated in a suitable culture medium for a sufficient time, in thatthis plucked hair follicle is brought into contact with a substancewhich is possibly active in the hair field, in that a label of theactivity of the said tested substance is quantitatively determined andin that the results of the quantitative determination are evaluated withrespect to a control.

The hair follicle used in the process according to the invention wasisolated by plucking. The plucking consists of a sudden separation ofthe hair follicle and of the dermis, generally carried out by a more orless strong pull exerted on the hair shaft of the follicle.

The plucked hair follicle can be intact, that is to say contain all theparts recognized by the person skilled in the art as constituting it(see, in this respect, "Science des traitements capillaires" Science ofHair Treatments!, Charles Zviak, published by Masson, 1987). Mentionwill be made, for example, and nonlimitingly, among these parts, of thedermal papillae, the hair bulb, the epithelial sheaths and the sebaceousgland. However, of course, the invention is not limited to the intactplucked hair follicle and also relates to any plucked hair folliclewhich, after isolation, would have retained only a portion of itsconstituent parts.

Plucking in order to isolate the hair follicle is a particularlyadvantageous procedure since it has the advantages of being noninvasiveand therefore nontraumatizing for the subject and of being simple andfast to carry out.

An additional advantage lies in the fact that plucking can be carriedout by the subject himself and anywhere, which places no additionalconstraint on the subject.

After isolation, the hair follicle is incubated in a suitable culturemedium possibly containing the substance to be tested. It is clearlyunderstood that the substance to be tested can be added to theincubation medium at any time, that is to say before or after bringingplucked hairs into contact with the said incubation medium. This medium,which is a nutrient medium, is at least composed of the componentsnecessary for keeping the hair follicle alive.

Of course, it can contain any other necessary component, for examplenecessary for the growth of the hair follicle as for example insulin,glutamine or hydrocortisone.

Mention may be made, by way of example, as culture medium well known tothe person skilled in the art, of Dulbecco's modified medium MEM,Williams' medium E, F12 medium, HAM medium or alternatively RPMI 1640,sold by the company Gibco-BRL, Biomed, Boehringer or alternativelySigma.

The incubation time is generally conditioned by the time necessary forthe hair follicle to respond to the substance which is possibly activein the hair field with which it is brought into contact, that is to saythe time necessary in order to see the appearance of a modification inthe level of expression of the label of the activity of the testedsubstance which is being quantitatively determined.

This incubation time can range from a few seconds to a number of days.By way of indication, the incubation time is generally between 5 secondsand 96 hours and preferably between 12 and 24 hours.

Label of the activity of the tested substance which is possibly activein the hair field is understood to mean, according to the invention, anycomponent, the presence, the absence, the modification in the expressionor the modification in the distribution of which can be measured inresponse to bringing the plucked hair follicle into contact with thesaid substance to be tested. Mention may be made, as an example of alabel, and without limitation, of protein, DNA, RNA, organelle, ion,metal, amino acid, lipid and liposoluble compound.

The activity of the substance to be tested is thus represented by thevariation in the label of the activity of the tested substance whichwill have been chosen to be quantitatively determined. Variation isunderstood to mean any modification in the amount, concentration ordistribution of the label which is quantitatively determined.

To do this, the process according to the invention comprises a stage ofquantitative determination of the label of the activity of the testedsubstance.

After incubation, this quantitative determination can be carried outdirectly on the culture medium for the components excreted by the cellor in the hair follicle for the non-excreted components.

Thus, and more particularly in the case where the component which isbeing searched for is not excreted, it is possible to envisage anadditional stage before quantitative determination, during which thehair follicle is ground, in order to make the label of the activity ofthe tested substance to be quantitatively determined more accessible.

Very clearly, whatever the embodiment of the process according to theinvention, any quantitative determination method known by the personskilled in the art can be used.

It is possible, by way of example and without limitation, to mention themethods of quantitative determination of proteins or of nucleic acids bycolorimetry, by electrophoresis, by reverse transcriptase andamplification by the chain polymerization technique, mass spectography,chromatography (in the gas phase or on a plate), immunological methods,or alternatively optical or electron microscopy for measuring the amountof an organelle.

The result of the quantitative determination, which represents thevariation in the label of the activity of the tested substance which hasbeen chosen to be quantitatively determined, cannot in itself bedirectly made use of. It only becomes advantageous in so far as it iscompared with the result of the same quantitative determination carriedout under the same conditions but without bringing the plucked hairfollicle into contact with the substance to be tested. Thus it is thatthe process according to the invention includes a stage during which anevaluation is carried out of the results of the quantitativedetermination with respect to a control.

The person skilled in the art easily determines, out of habit, thenature of the control necessary in the implementation of the process.

An advantage of the invention is that it provides, in the hair field, asimultaneously simple, fast and effective method for testing a substancewhich is possibly active which does not involve invasive stages.

In the hair field, the substances which are possibly active which it isdesired to test generally correspond to a modification in the state ofthe head of hair of the subject and preferentially the present or futurestate of this head of hair.

Generally, the substances to be tested possibly have an effect either onthe colour of the hair follicles or on the density, on the amount or onthe quality of the latter. These are substances which possibly have aneffect, for example, on the slowing down or halting of the growth, theloss or alternatively the intensified growth of the hair follicles.

Thus, the present invention makes it possible to test substances whichare possibly active in the hair field which have an effect on theslowing down or halting of the growth, the loss, the colouring oralternatively the intensified growth of the hair follicles.

The process according to the invention preferentially makes it possibleto test substances possibly having an effect on the slowing down orhalting of the growth, the loss or the colouring of the hair follicles.

As regards only the slowing down or halting of the growth or the loss ofthe hair follicles, the eventual consequence for the subject is a moreor less pronounced alopecia, which it is known can have aesthetic,psychological and social consequences for the affected subject.

More particularly, the process according to the invention makes itpossible to test substances possibly having an effect on the slowingdown or halting of the growth or the loss of the hair follicles.

The process according to the invention therefore makes it possible totest substances which are possibly active in the hair field in thetreatment of an alopecia.

The term alopecia covers a whole family of attacks on the hair folliclehaving the consequence, whatever the reason, of the partial or generaldefinitive loss of hair. Mention may be made, for example, of androgenicalopecia, alopecia areata (pelade) or alopecia totalis, or alternativelyalopecia universalis.

Without wishing to be bound by any one theory of the invention, it seemsthat some alopecias pass through at least one inflammatory stage.

An inflammatory stage of alopecia is characterized, inter alia, by amodification in the level of expression of mediators of inflammation.

Thus, the activity of a substance which is possibly active against atleast one inflammatory stage of an alopecia can thus be evaluated by theprocess according to the invention by quantitatively determining, in ahair follicle, at least one of the known mediators of inflammation,after incubation of the hair follicle with the said substance to betested.

Consequently, after incubation of the hair follicle with the substanceto be tested, the label of the activity of the said substance is, in theprocess according to the invention, advantageously one of the mediatorsof inflammation.

Mention may be made, among these mediators, of cytokines, including inparticular interleukin-1α, interleukin-1β, interleukin-6 or tumournecrosis factors α and β (TNF-α and -β), chemokines, such asinterleukin-8 or monocyte chemotactic and activating factor (MCAF), oralternatively other chemotactic factors responsible for the recruitmentof lymphocyte, monocyte, Langerhans or basophil cells at theinflammatory site, such as leukotrienes B₄, or alternatively otherfactors involved in the inflammatory cascade, such as arachidonic acidor prostaglandins, including in particular prostaglandins E₂.

The Applicant Company has indeed found that, in certain subjects, and inparticular in those exhibiting a beginning of alopecia, the level of theinterleukins is modified. The level of interleukin-1α and ofinterleukin-8, and more particularly the level of interleukin-1α, isincreased in the majority of subjects showing a beginning of alopecia.

The mediator of inflammation to be quantitatively determined in theprocess according to the invention is preferentially interleukin-1α andinterleukin-8 and more particularly the level of interleukin-1α.

The Applicant Company has also found that, in some subjects exhibitingan advanced alopecia, the level of prostaglandins E₂ is higher than inothers, suggesting an involvement of this mediator in the progression ofthis disorder. Thus, an early quantitative determination of anyvariation in the level of prostaglandins E₂ makes it possible toprognosticate a worsening of the alopecia. It is then possible tosuggest an appropriate treatment.

Thus, prostaglandins E₂ are another mediator of inflammation related toa hair disorder which the process according to the invention canquantitatively determine.

One of the advantages of the invention is thus to provide a simpleprocess for evaluating the activity of a substance which is possiblyactive against at least one inflammatory stage of an alopecia, whichsubstance could subsequently be used in the preparation of a cosmeticcomposition or of a medicament for the purpose of treating at least oneinflammatory stage of the alopecia.

Another advantage of the invention is to provide a simple means forevaluating the effectiveness of a treatment applied to a subject, byrepetitively and regularly making use of the process according to theinvention, from a hair follicle plucked from the said subject.

Moreover, the process according to the invention can make it possible toavoid a restrictive, cumbersome and expensive treatment of a specificsubject (for example, for whom the alopecia is not or is no longer inthe inflammatory stage) or when the test shows a response to this activeingredient in the form of a hyperproduction of inflammatory mediators(phenomena of allergy and/or of irritation).

Moreover, the process according to the invention makes it possible toevaluate the activity of a substance which can be used in thepreparation of a cosmetic composition or of a medicament for the purposeof treating at least one inflammatory stage of alopecia.

The Applicant Company has therefore shown, as will be seen in theexamples hereinbelow, that vitamin D and its derivatives, and moreparticularly 1,25-dihydroxyvitamin D₃, has an activity against at leastone of the mediators of inflammation related to alopecia.

A further subject of the invention is the use of the process accordingto the invention for evaluating the effectiveness of a treatment appliedto a subject.

Examples will now be given by way of illustration which should in no waylimit the scope of the invention.

EXAMPLE 1

Inhibiting effect of vitamin D₃ on the production of interleukin-1α,induced by interleukin-1β, in a plucked hair follicle.

In order to trigger a process mimicking an inflammatory stage in theplucked hair follicle, which represents the state of the follicle in asubject in the inflammatory stage of alopecia, at least one pluckedfollicle is incubated in the presence of interleukin-1β.

Ten plucked hairs are withdrawn from the region of the vertex of avolunteer. Five of these hairs are immediately incubated in Williams'medium E (sold by the company Gibco BRL), to which medium are addedantibiotics (penicillin G, 100 units/ml; streptomycin S, 100 μg/ml;amphotericin, 250 mg/ml) and interleukin-1β (recombinant marketed bySaxon Biochemicals GmbH) in the proportion of 100 ng/ml. The five otherhairs are incubated in the same medium (to which are added antibioticand interleukin-1β) in the presence of 1,25-dihydroxyvitamin D₃ (FranceBiochem) at the concentration of 0.1 nM.

After incubating for 20 hours, the culture supernatants are collected ina tube and then centrifuged for 5 minutes at 14000 revolutions/minute(Eppendorff centrifuge, model 5415C). The supernatants are thencollected in a clean tube and placed at 4° C. The concentration ofinterleukin-1α is then evaluated for 100 μl of supernatant using anELISA Biotrak kit marketed by the company Amersham, the recommendationsof the supplier being followed.

    ______________________________________    Concentration of IL-1α (in pg/ml)             Control Control + Vit. D.sub.3                                 % Inhibition    ______________________________________    Subject No. 1               3.8       2.1         44    Subject No. 2               80.7      40.5        50    Subject No. 3               13.8      8.8         36    Subject No. 4               34.7      28.9        17    Subject No. 5               18        12          33    ______________________________________

Statistical analyses were carried out by comparisons of the medians bycalculation of the Student's t (D. Schwartz: Methodes statistiques al'usage des medecins et des biologistes Statistical methods for the useof doctors and biologists!. Flammarion medecine et sciences, 1989).

The null hypothesis (H₀) is formulated as being: Control+Vit. D₃≧Control, in the context of a one-sided test, taking into account thefact that it concerns paired values.

According to Student, for the calculated t, there is rejection of H₀ atthe p=5% threshold. In this case, p=0.001.

Conclusion: Since H₀ is rejected, it may be concluded, at the 5%threshold, that the two populations (1: Control and 2: Control+Vit. D₃)differ significantly; the results obtained for the population 1 aresignificantly greater than those obtained with population 2.

The experiment shows that vitamin D₃ is a good inhibitor of theproduction of interleukin-1α, in a system reproducing the inflammatoryconditions of a hair follicle.

Consequently, this test can be used to evaluate the antiinflammatoryabilities of other analogues of vitamin D₃, by evaluation of theirability to inhibit the production of interleukin-1α.

EXAMPLE 2

Inhibiting effect of vitamin D₃ on the production of interleukin-8,induced by interleukin-1α, in a plucked hair follicle.

Fifteen hairs originating from volunteers are withdrawn from the regionof the nape of the neck. They are immediately incubated in Williams'medium E, marketed by the company Gibco BRL, to which are addedglutamine (2 mM) and antibiotics (penicillin G, 100 units/ml;streptomycin S, 100 μg/ml; amphotericin, 250 ng/ml), in the proportionof 200 μl of medium per plucked hair.

They are then divided into three batches: Batch No. 1: Hairs incubatedin the Williams' medium E mentioned above.

Batch No. 2: Hairs incubated in the Williams' medium E mentioned above,to which is added interleukin-1α (Biosource International) to a finalconcentration of 25 or 100 ng/ml (25 ng/ml for Subject No. 1 or 100ng/ml for Subjects No. 2 and 3).

Batch No. 3: Hairs incubated in the same medium as Batch No. 2, to whichis added 1,25-dihydroxyvitamin D₃ to 0.1 nM.

After 20 hours, the culture supernatants are collected in a tube andthen centrifuged for 5 minutes at 14000 revolutions/minute (Eppendorffcentrifuge, model 5415C). The supernatants are then collected in a cleantube and placed at 40° C.

The concentration of interleukin-8 is then evaluated for 100 μl ofsupernatant using an ELISA Biotrak kit marketed by the company Amersham,the instructions of the manufacturer being followed.

    ______________________________________           Batch No. 1                   Batch No. 2                             Batch No. 3           IL-8 (in pg/ml)     % Inhibition    ______________________________________    Subject No. 1             170       622       251     82    Subject No. 2             300       863       353     91    Subject No. 3             266       657       496     41    Subject No. 4             226       477       356     48    ______________________________________

Statistical analyses were carried out by comparisons of the means or ofthe medians by application of the Student's t test (D. Schwartz/Methodesstatistiques a l'usage des medecins et des biologistes Statisticalmethods for the use of doctors and biologists!. Flammarion medecine etsciences, 1989). The null hypothesis (H₀) is formulated as being: BatchNo. 3≧Batch No. 2, in the context of a one-sided test, taking intoaccount the fact that it concerns paired values.

According to Student, for the calculated t, there is rejection of H₀ atthe p=5% threshold. In this case, p=0.025.

Conclusion: Since H₀ is rejected, it may be concluded, at the 5%threshold, that the two batches differ significantly; the resultsobtained for Batch 2 are significantly greater than those obtained withBatch 3.

In these four cases, an inhibition of the production of interleukin-8 by1,25-dihydroxyvitamin D₃ is observed.

As IL-8 is an inflammatory chemokine induced by interleukin-1α, the testshows that 1,25-dihydroxyvitamin D₃, at 0.1 nM, has an antiinflammatoryeffect on the plucked follicle incubated in the presence ofinterleukin-1α. Consequently, this test can be used to evaluate theantiinflammatory abilities of other analogues of vitamin D₃, byevaluation of their ability to inhibit the production of interleukin-8.

EXAMPLE 3

Inhibiting effect of minoxidil on the production of prostaglandins E₂(PGE₂) in a hair follicle isolated by plucking.

10 hairs originating from three alopecic donors and from twonon-alopecic donors are withdrawn by plucking, from the region of thevertex. They are immediately divided into two batches: Batch No. 1: 5hairs incubated in Williams' medium E (marketed by the company GibcoBRL), to which are added glutamine (2 mM) and antibiotics (penicillin G,100 units/ml; streptomycin S, 100 μg/ml; amphotericin, 250 ng/ml).

Batch No. 2: 5 hairs incubated in the same medium as above butadditionally containing minoxidil at the final concentration of 10 μM.

After 18 hours, the plucked hairs from each group (5 hairs) arecollected in a microtube under an argon atmosphere and then stored at-80° C. On the day of the quantitative determination, 250 μl of degassedmethanol are added to each tube and then each sample is groundmechanically (10 rotations) using a pestle (tissue grind pestle SZ 20,marketed by the company Kontes). The ground material is then subjectedto ultrasound (20 pulses of 1 second; 50% amplitude) using a "Vibra cell72 434" ultrasonic device (marketed by the company Bioblock Scientific).The ground material which has been subjected to ultrasound iscentrifuged at 4° C. at 14000 revolutions/minute for 10 minutes(Eppendorff centrifuge, model 5415C). The supernatant is then collectedin a clean tube and lyophilized for one hour. The lyophilisate is takenup in 60 μl of phosphate buffer pH 7.5 supplied in the Biotrak kit(marketed by the company Amersham). The PGE₂ contents of 50 μl of thispreparation are then evaluated using the Biotrak kit according to themanufacturer's instructions.

    ______________________________________               Batch No. 1                        Batch No. 2                                   %               PGE.sub.2 in pg/ml                               Inhibition    ______________________________________    Donor #1 (alopecic)                 15.2       5.6        63    Donor #2 (alopecic)                 13.7       8.5        38    Donor #3 (alopecic)                 15.7       15.3       2.5    Donor #4 (non-alopecic)                 6.2        6.2        0    Donor #5 (non-alopecic)                 10.7       11.2       0    ______________________________________

These examples show that, in vitro, it is possible, by means of apharmacological agent which is supposed to promote hair regrowth in vivo(i.e. Minoxidil), to decrease the amount of PGE₂ produced by the pluckedhairs from certain alopecic individuals in the inflammatory stage(donors 1 and 2 in this example).

This observation can indicate a treatment with minoxidil in vivo inthese individuals who respond to minoxidil in vitro and, generally, thismethodology can indicate the choice of an active ingredient which isactive, with respect to the criterion studied (in this case, theproduction of PGE₂), for a specific donor. Conversely, this method ofevaluation makes it possible to avoid a restrictive, cumbersome andexpensive treatment for a specific subject (example of Donor 3) or whenthe test shows a response to this active ingredient in the form of ahyperproduction of inflammatory mediators (phenomena of allergy and/orof irritation).

What is claimed is:
 1. An assay for determining whether a substance ispotentially suitable for usage as an active ingredient in a hairtreatment composition which process comprises the following steps:(i)obtaining at least one plucked hair follicle from a subject; (ii)placing said at least one plucked hair follicle directly in a culturemedium containing nutrients, which culture medium contains a firstsubstance which is to be screened for its suitability as an activeingredient in a hair treating composition or to which culture mediumsaid first substance is added after the addition of said at least onehair follicle; (iii) incubating said at least one plucked hair folliclewith said culture medium containing said first substance which is beingscreened for its potential suitability as an active ingredient in a hairtreating composition for a sufficient amount of time for the productionof a second substance which mediates inflammation, the production ofwhich correlates to the potential suitability of said first substance asan active ingredient in a hair treating composition; (iv) quantitativelyassaying for the presence of said second substance which mediatesinflammation, and comparing these results to a control hair folliclecontaining composition which does not contain the first substance; and(v) determining based on the results of said assay the potentialsuitability of said first substance as an active ingredient in a hairtreating composition.
 2. The assay of claim 1, wherein the culturemedium maintains the viability of said at least one hair follicle for asufficient time to conduct the assay.
 3. The assay of claim 1, whereinthe culture medium contains at least one substance which provides forthe growth of said at least one hair follicle.
 4. The assay of claim 1,wherein the incubating step (iii) is effected for a time period rangingfrom about 5 seconds to 96 hours.
 5. The assay of claim 4, wherein saidtime period ranges from about 12 to about 24 hours.
 6. The assay ofclaim 1, wherein assay step (iv) measures the concentration, amount ordistribution of said second substance in the culture medium or in thehair follicle.
 7. The assay of claim 1, wherein the assay step (iv)assays the presence of said second substance which is contained in thehair follicle.
 8. The assay of claim 7, wherein the incubated hairfollicle is ground prior to conducting the assay step (iv).
 9. The assayof claim 1, wherein the screened first substance is selected from thegroup consisting of nucleic acids, proteins, combinations of differentproteins which may be separate or bound to one another, ions, cellorganelles, lipids and polysaccharides.
 10. The assay of claim 1 whereinthe first substance which is being screened for usage as an activeingredient in hair treating compositions putatively affects at least oneof the following hair follicle properties; (i) coloration, (ii)survival, (iii) hair loss, (iv) retardation of growth and (iv) enhancingof growth.
 11. The assay of claim 1, which is used to identify asubstance potentially suitable for treatment of alopecia.
 12. The assayof claim 11, wherein alopecia is selected from the group consisting ofandrogenic alopecia, alopecia areata and alopecia totalis.
 13. The assayof claim 1, wherein said assayed second substance is selected from thegroup consisting of cytokines, chemokines, chemotactic factorsresponsible for the recruitment of lymphocytes, monocytes, Langerhanscells, and basophils to sites of inflammation, and other factorsinvolved in the inflammatory cascade.
 14. The assay of claim 1, whereinsaid assayed second substance is selected from the group consisting ofinterleukin-1α, interleukin-1β, interleukin-6, tumor necrosis factors αand β, interleukin-8, monocyte chemotactic and activating factor (MCAF),leukotrienes B₄, arachidonic acid and prostaglandins.
 15. The assay ofclaims 14, wherein the assayed prostaglandin is prostaglandin E₂. 16.The assay of claim 11, wherein said assayed second substance is selectedfrom the group consisting of cytokines, chemokines, chemotactic factorsresponsible for the recruitment of lymphocytes, monocytes, Langerhanscells, and basophils to sites of inflammation, and other factorsinvolved in the inflammatory cascade.
 17. The assay of claim 11, whereinsaid assayed second substance is selected from the group consisting ofinterleukin-1α, interleukin-1β, interleukin-6, tumor necrosis factors αand β, interleukin-8, monocyte chemotactic and activating factor,leukotrienes B₄, arachidonic acid and prostaglandins.
 18. The assay ofclaim 1, wherein said assayed second substance is an interleukin. 19.The assay of claim 11, wherein said assayed second substance is aninterleukin.
 20. The assay of claim 12, wherein the interleukin isselected from the group consisting of interleukin-1α, interleukin-1β,interleukin-6 and interleukin-8.
 21. The assay of claim 19, wherein theinterleukin is selected from the group consisting of interleukin-1α,interleukin-1β, interleukin-6 and interleukin-8.
 22. The assay of claim20, wherein the assayed interleukin is interleukin-1α.
 23. The assay ofclaim 20, wherein the assayed interleukin is interleukin-8.
 24. Theassay of claim 21, wherein the assayed interleukin is interleukin-1α.25. The assay of claim 21, wherein the assayed interleukin isinterleukin-8.
 26. The assay of claim 1, wherein the assayed secondsubstance is a prostaglandin.
 27. The assay of claim 26, wherein theassayed prostaglandin is a prostaglandin E₂.
 28. The assay of claim 1,wherein the first substance screened for its suitability as an activeagent in hair treating compositions is a substance potentially suitablefor treating and/or preventing an inflammatory stage of alopecia.